High-voltage-activated calcium (CaV1/CaV2) channels are necessary for the function of excitable cells. Molecules that inhibit CaV1/CaV2 channels powerfully regulate physiology, and are important or potential therapeutics for many serious diseases including: hypertension, neuropathic pain, cardiac arrhythmias, and Parkinson's disease. CaV1/CaV2 channels are potently inhibited by a four-member family of monomeric G- proteins known as RGK (Rad, Rem, Rem2, Gem/Kir) proteins. RGKs are expressed in excitable tissues, and their expression level often changes correlatively with disease, suggesting their strong regulation of CaV1/CaV2 has broad patho-physiological implications. Engineered RGKs have potential therapeutic applications as genetically-encoded CaV channel blockers (CCBs) for a broad range of diseases. For specific applications, genetically encoded inhibitors may provide a higher therapeutic index than traditional small molecule CCBs because they can be locally expressed, thereby achieving effective CaV channel block while minimizing off- target effects. The precise molecular mechanisms by which RGKs inhibit CaV1/CaV2 channels are not well- understood. Our preliminary data hint at a surprising degree of customization and complexity where distinct RGK proteins differentially use multiple mechanisms and structural determinants to inhibit individual CaV1/CaV2 channel isoforms. Precise understanding of the mechanisms underlying customized RGK inhibition of CaV1/CaV2 channels is critical for insights into the patho-physiological ramifications of this channel regulation, as well as efforts to engineer useful new genetically-encoded CCBs. Our long-term objective is to furnish fundamental understanding of the diverse molecular mechanisms and structural determinants underlying RGK inhibition of CaV1/CaV2 channels and to bridge these insights to: (i) a new appreciation of the patho-physiological implications of this channel modulation; and (ii) the design of novel, useful genetically-encoded CCBs as potential therapeutics. We combine whole-cell and single-channel electrophysiology, fluorescence resonance energy transfer (FRET), molecular biology, channel engineering, and biochemical approaches to address three specific Aims: (1) Dissect mechanisms the RGK protein, Rem, uses to inhibit recombinant CaV1.2 channels. (2) Determine and contrast mechanisms of RGK inhibition across the CaV1/CaV2 channel family. (3) Dissect mechanisms of RGK inhibition of native CaV1.2 channels in cardiomyocytes.